This is a second revision of an RO1 proposal [original score 183 (20.4 percent); 1st revision score 160 (24.5 percent)] to determine mechanisms of action of a murine gamma-herpesvirus 68 (gammaHV68) encoded member of the Regulators of Complement Activation (RCA) family of proteins. Using the genetically manipulable gammaHV68 virus in mouse strains carrying null mutations in complement proteins (C3-/- or Factor B-/-) or antibody (B cell deficient), we will determine how the gammaHV68 RCA protein functions in vitro and in vivo in the natural host. Analysis of the mechanisms by which this RCA protein functions provides a window upon the broader question of how complement +/-antibody controls or promotes viral infection and virus- associated inflammation. Little is known about how avoidance of complement via viral RCA proteins may alter pathogenesis, latency, or inflammation in organs such as lung, spleen, and the great elastic arteries. RCA proteins contain multiple repeats of an approximately 60 amino acid motif called the short consensus repeat (SCR). On sequencing the genome of gammaHV68, we discovered that gene 4 encodes an RCA protein homolog with 4 SCRs. This protein is conserved in both position and structure between Kaposi's sarcoma herpesvirus (KSHV, HHV8), herpesvirus saimiri, and gammaHV68, strongly arguing for an important function. We have shown that gammaHV68 gene 4 encodes both membrane bound and soluble forms of the gammaHV68 RCA protein, and that the gammaHV68 RCA protein inhibits both classical and alternative pathways of complement activation. We have isolated a mutant gammaHV68 lacking the gammaHV68 RCA protein that grows normally fibroblasts, validating genetic approaches to these mechanistic questions. Our Aims are: Aim 1) Characterization of transcripts and encoded proteins arising from gammaHV68 gene 4; Aim 2) Define the mechanism(s) by which the gammaHV68 protein inhibits complement activation; Aim 3) Define the role of the gammaHV68 RCA protein in gammaHV68 pathogenesis. The 3 PIs committed to these studies have expertise in viral pathogenesis and immunology, molecular virology, and the biochemistry of complement. These investigators have the enthusiasm and expertise to perform the integrated virologic, molecular, biochemical, and pathogenetic studies necessary to fully elucidate molecular mechanisms of gamma-herpesvirus RCA protein function in vitro and in vivo.